Methods and compositions for producing plants with elevated brix

ABSTRACT

The invention provides compositions and methods relating to the production of  Cucumis melo  plants with elevated Brix and/or enhanced fruit color. The invention further provides plants, plant parts, and seeds comprising such elevated Brix. The invention allows introgression of the identified high Brix QTL in an desired genetic background of a sexually compatible plant species.

This application claims the priority of U.S. Provisional Appl. Ser. No.61/642,978, filed May 4, 2012, the entire disclosure of which isincorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“SEMB005US_ST25.txt”, which is 39 kilobytes as measured in MicrosoftWindows operating system and was created on May 1, 2013, is filedelectronically herewith and incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the field of plant breeding and, morespecifically, to methods and compositions for producing melon plantswith elevated Brix.

Description of Related Art

Melon fruits are highly appreciated worldwide and are often eaten as afresh product. Melons are members of the gourd family (Cucurbitaceae), aclass of trailing annual vines that also includes squash, pumpkin andcucumber. They have large broad leaves, stems covered in light pricklesand small yellow flowers. The fruit themselves are soft fleshed with acentral cavern containing seeds, surrounded by a thick protective rind.

Taxonomically, melons are broadly divided into two groups: watermelons(species Citrullus lanatus) and muskmelons (species Cucumis melo L.). C.melo includes a wide variety of cultivars producing fruits of differentshape, external appearance and flesh color, including such melons asCanary, Cantaloupe (including Western Shipper, North American andCharentais types), Casaba, Hami, Honeydew, Navajo Yellow, Piel de Sapo,Santa Claus, Sugar melon, Ambrosia, Bailan, Galia, Ogen, Persian, andSharlyn.

One important goal of melon breeding is to combine various desirabletraits in a single variety/hybrid. While breeding efforts to date haveprovided a number of useful melon lines and varieties with beneficialtraits, there remains a need in the art for new lines and varieties withfurther improved traits and methods for their production. In many cases,such efforts have been hampered by difficulties in identifying and usingalleles conferring beneficial traits.

SUMMARY OF THE INVENTION

The present invention overcomes limitations in the prior art byproviding a Cucumis melo plant comprising at least a first introgressedchromosomal region conferring increased Brix content relative to a plantlacking the region, wherein the region is selected from the groupconsisting of a high Brix content contributing QTL defined by flankingmarkers NU0219671 and NU0218915 on Cucumis melo linkage group 4, a highBrix content contributing QTL defined by flanking markers NU0220114 andNU0219142 on Cucumis melo linkage group 10, and high Brix contentcontributing QTL defined by flanking markers NU0220114 and NU0220323 onCucumis melo linkage group 10. Thus in certain embodiments the Cucumismelo plant comprises said high Brix content contributing QTL defined byflanking markers NU0219671 and NU0218915 and said high Brix contentcontributing QTL defined by flanking markers NU0220114 and NU0219142. Inyet other embodiments, the Cucumis melo plant comprises said high Brixcontent contributing QTL defined by flanking markers NU0219671 andNU0218915 and said high Brix content contributing QTL defined byflanking markers NU0220114 and NU0220323.

In particular embodiments, the chromosomal region conferring increasedBrix comprises the QTL found in Cucumis melo line BEST, a sample of seedof Cucumis melo line BEST having been deposited under ATCC AccessionNumber PTA-12263. The Cucumis melo plant may be further defined as oneof the market class group consisting of Piel de Sapo, Juan Canary,Amarillo, Earl's Type, Honeydew, Orange-flesh honeydew, Hami Melon,Crenshaw, Casaba, Ananas, Galia, Charentais, Italian-type, and WesternShipper.

in some embodiments the Cucumis melo plant is homozygous for saidchromosomal region. In other embodiments the plant produces fruit withan average Brix content of at least 9° Brix at fruit maturity. Incertain embodiments the chromosomal region confers an increase of atleast 1° Brix relative to an otherwise essentially isogenic plantlacking the chromosomal region. In additional embodiments the plant isinbred. In other embodiments the plant is hybrid.

In another aspect, the invention provides a seed of the Cucumis meloplant. In yet another aspect, the invention provides a part of such aplant. In some embodiments, the plant part is selected from the groupconsisting of a leaf, pollen, an ovule, a fruit, rootstock, a scion, anda cell. In a particular embodiment the plant part is a fruit.

A tissue culture of regenerable cells of the plant is also an aspect ofthe invention. In one embodiment the tissue culture comprises cells orprotoplasts from a plant part selected from the group consisting of anembryo, a meristem, a cotyledon, pollen, a leaf, anthers, a root, a roottip, pistil, flower, seed and a stalk.

Another aspect of the present invention is a method of obtaining Cucumismelo germplasm comprising the steps of: a) assaying Cucumis melo plantsor a seed thereof for the presence of at least a first genetic markergenetically linked to a chromosomal region conferring increased Brixcontent relative to a plant lacking the region, wherein the region isselected from the group consisting of a high Brix content contributingQTL defined by flanking markers NU0219671 and NU0218915 on Cucumis melolinkage group 4 and a high Brix content contributing QTL defined byflanking markers NU0220114 and NU0219142 on Cucumis melo linkage group10; and b) selecting at least a first Cucumis melo plant or seed thereofcomprising the genetic marker and the QTL that confers increased Brixcontent. In an embodiment of the method, the high Brix contentcontributing QTL on Cucumis melo linkage group 10 is defined by flankingmarkers NU0220114 and NU0220323. In certain embodiments, a high Brixcontent contributing allele has been inherited from Cucumis melo lineBEST, or a progeny of any generation thereof comprising said allele fromCucumis melo line BEST, a sample of seed of said melon line BEST havingbeen deposited under ATCC Accession Number PTA-12263.

In certain embodiments the first genetic marker is selected from thegroup consisting of NU0219671, NU0221090, NU0218604, NU0218684,NU0218148, NU0243432, NU0243324, NU0219095, NU0218257, NU0219354,NU0219672, NU0243607, NU0219118, NU0220372, NU0219774, NU0219889,NU0244419, NU0244478, NU0220446, NU0219650, NU0244718, NU0218943,NU0218512, NU0219136, NU0218860, NU0220597, NU0219475, NU0243542,NU0220825, NU0219448, NU0243629, NU0218829, NU0218923, NU0243807,NU0244022, NU0218871, NU0218901, NU0220173, NU0220684, NU0218887,NU0219803, NU0219004, NU0219642, NU0219025, NU0218757, NU0219015,NU0219386, NU0219676, NU0243281, NU0243478, NU0219177, NU0219316,NU0219426, NU0220839, NU0244508, NU0244665, NU0218187, NU0218513,NU0221036, NU0243535, NU0220613, NU0218306, NU0218509, NU0219086,NU0220451, NU0244507, NU0219425, NU0244254, NU0220942, NU0218848,NU0244041, NU0220202, NU0220577, and NU0218915.

In particular embodiments the first genetic marker is selected from thegroup consisting of NU0219671, NU0243432, NU0219672, NU0219774,NU0220446, NU0218512, NU0243542, NU0219676, NU0220613, NU0244254,NU0220202, and NU0218915. In certain embodiments of the method, thefirst genetic marker is selected from the group consisting of NU0219671and NU0218915.

In some embodiments, the first genetic marker is selected from the groupconsisting of NU0220114, NU0219003, NU0219125, NU0220581, NU0220762,NU0220116, NU0220849, NU0218843, NU0220323, NU0220985, NU0244737,NU0219214, NU0218908, NU0244415, NU0220766, NU0243268, NU0218664,NU0219506, NU0220650, NU0219176, NU0219728, NU0244427, NU0219075,NU0243568, NU0220301, NU0243578, NU0218206, NU0218955, NU0219589,NU0221079, NU0219683, NU0219740, NU0218191, NU0219782, and NU0219142. Incertain embodiments the first genetic marker is selected from the groupconsisting of NU0220114, NU0219125, NU0220323, NU0244737, NU0218664,NU0243568, NU0219683, and NU0219142. In particular embodiments the firstgenetic marker is selected from the group consisting of NU0220114 andNU0219142. In other embodiments, the first genetic marker is selectedfrom the group consisting of NU0220114 and NU0220323.

In other embodiments, the method comprises assaying the Cucumis meloplants or a seed thereof for a high Brix content contributing QTLdefined by flanking markers NU0219671 and NU0218915 on Cucumis melolinkage group 4 and a high Brix content contributing QTL defined byflanking markers NU0220114 and NU0219142 on Cucumis melo linkage group10. In certain embodiments, the method comprises assaying the Cucumismelo plants or a seed thereof for a high Brix content contributing QTLdefined by flanking markers NU0219671 and NU0218915 on Cucumis melolinkage group 4 and a high Brix content contributing QTL defined byflanking markers NU0220114 and NU0220323 on Cucumis melo linkage group10. In some embodiments assaying the Cucumis melo plants may comprisePCR, single strand conformational polymorphism analysis, denaturinggradient gel electrophoresis, cleavage fragment length polymorphismanalysis, TAQMAN assay, and/or DNA sequencing.

Another aspect of the invention is a method of Cucumis melo plantbreeding comprising: a) assaying Cucumis melo plants or seeds thatproduce the plants for the presence of at least a first genetic markergenetically linked to a chromosomal region conferring increased Brixcontent relative to a plant lacking the region, wherein the region is ahigh Brix content contributing QTL defined by flanking markers NU0219671and NU0218915 on Cucumis melo linkage group 4, or a high Brix contentcontributing QTL defined by flanking markers NU0220114 and NU0219142 onCucumis melo linkage group 10; and b) selecting at least a first Cucumismelo plant or seed that produces the plant comprising the genetic markerand the QTL that confers increased Brix; and c) crossing the firstCucumis melo plant with itself or a second Cucumis melo plant to produceprogeny Cucumis melo plants comprising the QTL that confers increasedBrix.

In some embodiments of this method, the first genetic marker is selectedfrom the group consisting of NU0219671, NU0221090, NU0218604, NU0218684,NU0218148, NU0243432, NU0243324, NI0219095, NU0218257, NU0219354,NU0219672, NU0243607, NU0219118, NU0220372, NU0219774, NU0219889,NU0244419, NU0244478, NU0220446, NU0219650, NU0244718, NU0218943,NU0218512, NU0219136, NU0218860, NU0220597, NU0219475, NU0243542,NU0220825, NU0219448, NU0243629, NU0218829, NU0218923, NU0243807,NU0244022, NU0218871, NU0218901, NU0220173, NU0220684, NU0218887,NU0219803, NU0219004, NU0219642, NU0219025, NU0218757, NU0219015,NU0219386, NU0219676, NU0243281, NU0243478, NU0219177, NU0219316,NU0219426, NU0220839, NU0244508, NU0244665, NU0218187, NU0218513,NU0221036, NU0243535, NU0220613, NU0218306, NU0218509, NU0219086,NU0220451, NU0244507, NU0219425, NU0244254, NU0220942, NU0218848,NU0244041, NU0220202, NU0220577, and NU0218915. In other embodiments,the first genetic marker is selected from the group consisting ofNU0220114, NU0219003, NU0219125, NU0220581, NU0220762, NU0220116,NU0220849, NU0218843, NU0220323, NU0220985, NU0244737, NU0219214,NU0218908, NU0244415, NU0220766, NU0243268, NU0218664, NU0219506,NU0220650, NU0219176, NU0219728, NU0244427, NU0219075, NU0243568,NU0220301, NU0243578, NU0218206, NU0218955, NU0219589, NU0221079,NU0219683, NU0219740, NU0218191, NU0219782, and NU0219142.

The method may further comprise the step of: d) selecting a progenyplant comprising the QTL and crossing the progeny plant with itself or athird Cucumis melo plant to produce additional progeny plants. Incertain embodiments of the method, step (d) may be repeated about 2-10times. Repeating step (d) may further comprise, in at least onegeneration, assaying Cucumis melo progeny plants or seed thereof for thepresence of a genetic marker genetically linked to said QTL.

In some embodiments, selecting the first Cucumis melo plant comprisesselecting the plant based on the presence of a genetic marker selectedfrom the group consisting of NU0219671, NU0243432, NU0219672, NU0219774,NU0220446, NU0218512, NU0243542, NU0219676, NU0220613, NU0244254,NU0220202, NU0218915, NU0220114, NU0219125, NU0220323, NU0244737,NU0218664, NU0243568, NU0219683, and NU0219142. In other embodiments thechromosomal region conferring increased Brix comprises a high Brix QTLfound in Cucumis melo line BEST, wherein the QTL is defined by flankingmarkers NU0219671 and NU0218915 on Cucumis melo linkage group 4 or byflanking markers NU0220114 and NU0219142 on Cucumis melo linkage group10; and wherein a sample of seed of melon line BEST has been depositedunder ATCC Accession Number PTA-12263. In another embodiment, assayingthe Cucumis melo plants comprises PCR, single strand conformationalpolymorphism analysis, denaturing gradient gel electrophoresis, cleavagefragment length polymorphism analysis, TAQMAN assay, and/or DNAsequencing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Brix levels of the WSH controls and candidate Brix donor linesA-E, grown in two locations, with standard errors indicated.

FIG. 2: Average Brix levels of the parents and test cross progeny of WSH39-1046 AN with candidate Brix donor lines A, C, and D, with standarderrors indicated.

FIG. 3: Histogram of the average Brix levels of the F3 families derivedfrom BEST, with the number of families in each bin on the y-axis, andbins labeled with the high end values.

FIG. 4: Shown are the log of the odds (LOD) plots for linkage groups 4and 10. The graphs represent raw and adjusted Brix values from threetrial locations. The horizontal line indicates the permutation derived95% confidence level for the presence of a QTL at any given locus. ThecM units are the map positions on the melon consensus map.

FIG. 5: The positions of the two Brix QTL on the Cucumis melo consensusgenetic map are illustrated with thick bars indicating the 95%confidence interval. The QTL on linkage group 4 (“LG4”) is bounded bymarkers NU0219671 (map position 42.9067 cM) and NU0218915 (map position63.5466 cM), while the QTL on LG10 is bounded by markers NU0220114 (mapposition 1.670227 cM) and NU0219142 (map position 14.3257 cM). Forclarity, only regions of interest are shown (from ˜37.9 to ˜67.3 cM onlinkage group 4, and 0.0 to ˜40.3 cM on LG10).

FIG. 6: Mean Brix through fruit development in high Brix sources. Datacolumns at each timepoint for Days Post Anthesis on the X axis are fromleft to right: High Brix Line 1, WSH39 1046AN, High Brix Line 2, Best,and High Brix Line 3, Note there is no data for High Brix Line 1 at 42Days Post Anthesis.

FIG. 7: LS mean values of Brix for each line and time point postanthesis, Y axis is LS means Brix and X axis is days post anthesis(dpa). ‘Best’ is shown in black, Q4:B in dark gray, Q4:1046 in mediumgray, and WSH 39-1046 AN in light gray. The approximate date of slip isindicated by the arrows. Note that the process of slipping progressesfrom quarter slip through to full slip, with quarter slip representingcommercial maturity. Since Q4:B was at full slip at 34 DPA we can inferthat quarter slip would occur approximately 1 day earlier. Q4:1046 wasat quarter slip at 37 DPA. WSH 39-1046 AN does not slip untilapproximately 42 DPA, Q4:B registers as statistically different thanQ4:1046 with Student's t at P<0.05 for 28, 31, and 34 dpa.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to plants,seeds and derivatives of Cucumis melo L, comprising introgressedchromosomal regions conferring elevated Brix. In particular, twointrogressed QTL from melon line BEST are disclosed, and methods andcompositions allowing for efficient marker-assisted selection to achieveprogeny hybrid melon plants displaying enhanced fruit sweetness areprovided. Two QTL were identified from donor line BEST which conferincreased Brix. The QTL on LG4 is bounded by markers NU0219671 (mapposition 42.9067 cM) and NU0218915 (map position 63.5466 cM), while theQTL on LG10 is bounded by markers NU0220114 (map position 1.670227 cM)and NU0219142 (map position 14.3257 cM), including the region bounded bymarkers NU0220114 (map position 1.670227 cM) and NU0220323 (map position4.48 cM). Progeny lines derived from line BEST displayed significantlyelevated Brix levels as compared with parental and control lines infield trials performed at multiple growing locations.

Identification of these QTL allows for efficient marker-assistedselection of progeny melon lines for enhanced sweetness, i.e. elevatedBrix, as well as deeper and/or earlier developing fruit color. Aclassical phenotypic breeding approach to select for such traits wouldrequire large replicated field trials in appropriate environments withextensive sampling of fruit, necessitating significant use of space,time, and labor, and could result in reduced ability to select othertraits of importance to a breeding program, such as for diseaseresistance or other desired trait(s). Selection for Brix QTL withmolecular marker tests on seedlings thus allows for large gains inbreeding efficiency. These QTL may he introgressed into parental inbredlines to provide improved fruit quality in melons, for instance as grownin Central and South America for export to the US or elsewhere. Theavailability of high quality fruits with improved sweetness can lead toenhanced marketability and increased consumption, also improving humannutrition.

In particular embodiments, the invention provides markers geneticallylinked to the described QTL for Brix content which are located onCucumis melo and LG10. In certain embodiments, the markers are within 20cM, 10 cM, 5 cM, 3 cM, 1 cM, or less, of a QTL on melon LG4 or LG10 thatallow for enhanced Brix. The presence of a given marker may beidentified by use of well known techniques, such as by nucleic aciddetection methods utilizing probes or primers comprising a sequence asdisclosed herein. Genetic map position, physical map position, andallele and sequence information are given for selected genetic markerslinked to QTL on LG4 and LG10 in Table 1 (SEQ ID NOs:1-110). For markerNU0218908, the estimated genetic map position (e.g. location relative tonearby markers) may be inaccurately estimated.

TABLE 1 Map position and allele information for selected genetic markerson melon LG4 and LG10 (SEQ ID NOs: 1-110); “n/a” = not available.Linkage Unfavorable Group Genetic map Physical map Favorableallele_WSH-39- SEQ Marker (“LG”) position (cM) position (bp);allele_BEST 1046-AN ID NO NU0219671 4 42.90670132 9258434 CC AA 1NU0221090 4 42.90670132 9270762 2 NU0218604 4 43.0979374 9427935 3NU0218684 4 43.23036242 9616649 4 NU0218148 4 44.23479447 9854150 5NU0243432 4 44.23479447 9965073 GG AA 6 NU0243324 4 44.79668663 106630977 NU0219095 4 45.17459331 10798547 8 NU0218257 4 45.20843312 10820633 9NU0219354 4 45.61944747 n/a 10 NU0219672 4 45.7377921 11393641 CC TT 11NU0243607 4 45.89414062 11394205 12 NU0219118 4 47.56222556 13409958 13NU0220372 4 47.56222556 11920721 14 NU0219774 4 49.07916075 13738760 CCGG 15 NU0219889 4 49.92630366 13741661 16 NU0244419 4 49.9263036614020679 17 NU0244478 4 49.92630366 14025383 18 NU0220446 4 50.1575022314314960 TT AA 19 NU0219650 4 50.41420068 14393543 20 NU0244718 450.41420068 14391032 21 NU0218943 4 51.44499499 15014313 22 NU0218512 452.56649468 15436556 TT AA 23 NU0219136 4 52.56649468 15661055 24NU0218860 4 53.42270279 15770285 25 NU0220597 4 53.4428944 15948257 26NU0219475 4 53.45581073 16981336 27 NU0243542 4 53.45581073 17307866 GGAA 28 NU0220825 4 53.45581073 16554223 29 NU0219448 4 53.4558107317207239 30 NU0243629 4 53.45581073 n/a 31 NU0218829 4 53.4640582117298733 32 NU0218923 4 53.46405821 17297098 33 NU0243807 4 53.4640582116342905 34 NU0244022 4 53.46405821 16341935 35 NU0218871 4 53.4702731617208693 36 NU0218901 4 53.55065617 16545065 37 NU0220173 4 53.5541547616337772 38 NU0220684 4 53.62195987 15899732 39 NU0218887 4 53.6408638916752167 40 NU0219803 4 53.88873817 16753311 41 NU0219004 4 54.0046105817341552 42 NU0219642 4 54.00461058 n/a 43 NU0219025 4 54.2081224217536806 44 NU0218757 4 54.31894245 17540043 45 NU0219015 4 54.3189424517342789 46 NU0219386 4 54.45681139 18719229 47 NU0219676 4 54.51656607n/a AA GG 48 NU0243281 4 54.51656607 18987560 49 NU0243478 4 54.5165660719132953 50 NU0219177 4 54.51656607 19521311 51 NU0219316 4 55.2813499921180382 52 NU0219426 4 55.28134999 21327981 53 NU0220839 4 55.2813499921092239 54 NU0244508 4 55.28134999 21088377 55 NU0244665 4 55.2813499921180722 56 NU0218187 4 55.28134999 21196877 57 NU0218513 4 55.2813499920650737 58 NU0221036 4 55.28134999 20644745 59 NU0243535 4 55.2813499921329207 60 NU0220613 4 55.8264394 21436236 AA GG 61 NU0218306 455.97274243 21568222 62 NU0218509 4 55.97274243 21587099 63 NU0219086 455.97274243 21558389 64 NU0220451 4 55.97274243 21561207 65 NU0244507 455.97274243 21662407 66 NU0219425 4 58.92886945 21696482 67 NU0244254 459.83874887 22253203 CC TT 68 NU0220942 4 59.87520725 22510081 69NU0218848 4 60.31911906 22835218 70 NU0244041 4 60.41467622 24081543 71NU0220202 4 61.86643091 n/a TT GG 72 NU0220577 4 63.48790382 24558770 73NU0218915 4 63.54660949 24769784 GG AA 74 NU0220114 10 1.670227256383862 CC AA 75 NU0219003 10 1.825624361 494829 76 NU0219125 102.460949526 537783 TT CC 77 NU0220581 10 2.529190171 555635 78 NU022076210 3.802854385 659817 79 NU0220116 10 4.140708555 671203 80 NU0220849 104.140708555 668944 81 NU0218843 10 4.47813079 762077 82 NU0220323 104.47813079 735937 GG TT 83 NU0220985 10 5.74160713 838303 84 NU024473710 5.74160713 839213 AA GG 85 NU0219214 10 5.74160713 870269 86NU0218908 10 5.74160713 1012622 87 NU0244415 10 5.958658859 888018 88NU0220766 10 7.209687724 1039711 89 NU0243268 10 7.209687724 1054026 90NU0218664 10 7.52970735 1111402 TT AA 91 NU0219506 10 7.9509439361151696 92 NU0220650 10 9.416408629 1239143 93 NU0219176 10 9.4187162151233075 94 NU0219728 10 9.668938423 1282101 95 NU0244427 10 9.6689384231261013 96 NU0219075 10 10.44636116 1338918 97 NU0243568 10 10.446361161380818 TT CC 98 NU0220301 10 10.89127599 1420487 99 NU0243578 1010.98050134 1421936 100 NU0218206 10 11.81903381 1465614 101 NU021895510 11.81903381 1463213 102 NU0219589 10 11.81903381 1391537 103NU0221079 10 12.41050087 1499072 104 NU0219683 10 12.60551646 1556982 TTCC 105 NU0219740 10 12.60551646 1557750 106 NU0218191 10 12.605516461604728 107 NU0219782 10 14.28348493 1671415 108 NU0219142 10 14.32570251676663 TT AA 109 NU0218502 10 18.82 n/a GG AA 110

In some embodiments, the markers used to follow the presence of any ofthese QTL of LG4 and/or LG10 are selected from the group consisting of:NU0219671, NU0218915, NU0220114, and NU0219142.

In further embodiments, the markers used to follow the presence of anyof these QTL are selected from the group consisting of: NU0219671,NU0243432, NU0219672, NU0219774, NU0220446, NU0218512, NU0243542,NU0219676, NU0220613, NU0244254, NU0220202, and NU0218915. In otherembodiments the markers used to follow the presence of any of these QTLare selected from the group consisting of: NU0220114, NU0219125,NU0220323, NU0244737, NU0218664, NU0243568, NU0219683, and NU0219142.

In yet other embodiments, the markers used to follow the presence of anyof these QTL are selected from the group consisting of: NU0219671,NU0221090, NU0218604, NU0218684, NU0218148, NU0243432, NU0243324,NU0219095, NU0218257, NU0219354, NU0219672, NU0243607, NU0219118,NU0220372, NU0219774, NU0219889, NU0244419, NU0244478, NU0220446,NU0219650, NU0244718, NU0218943, NU0218512, NU0219136, NU0218860,NU0220597, NU0219475, NU0243542, NU0220825, NU0219448, NU0243629,NU0218829, NU0218923, NU0243807, NU0244022, NU0218871, NU0218901,NU0220173, NU0220684, NU0218887, NU0219803, NU0219004, NU0219642,NU0219025, NU0218757, NU0219015, NU0219386, NU0219676, NU0243281,NU0243478, NU0219177, NU0219316, NU0219426, NU0220839, NU0244508,NU0244665, NU0218187, NU0218513, NU0221036, NU0243535, NU0220613,NU0218306, NU0218509, NU0219086, NU0220451, NU0244507, NU0219425,NU0244254, NU0220942, NU0218848, NU0244041, NU0220202, NU0220577, andNU0218915.

In still yet other embodiments, the markers used to follow the presenceof any of these QTL are selected from the group consisting of:NU0220114, NU0219003, NU0219125, NU0220581, NU0220762, NU0220116,NU0220849, NU0218843, NU0220323, NU0220985, NU0244737, NU0219214,NU0218908, NU0244415, NU0220766, NU0243268, NU0218664, NU0219506,NU0220650, NU0219176, NU0219728, NU0244427, NU0219075, NU0243568,NU0220301, NU0243578, NU0218206, NU0218955, NU0219589, NU0221079,NU0219683, NU029740, NU0218191, NU0219782, and NU0219142.

In still further embodiments, the invention provides a method comprisingobtaining a progeny plant that comprises a(n) allele(s) specifyingincreased Brix by identifying one or more genetic markers geneticallylinked to a high Brix melon QTL. Identifying the genetic markers maycomprise a phenotypic, a genetic, or a biochemical test, and may includescreening a parent and/or progeny plant for the presence of one or moreof the alleles described herein, including, for example, one or morealleles of markers on melon linkage group 4, such as markers NU0219671,NU0243432, NU0219672, NU0219774, NU0220446, NU0218512, NU0243542,NU0219676, NU0220613, NU0244254, NU0220202, and NU0218915, among others.In some embodiments, screening for an allele of a marker linked within10 cM of any of these above listed markers is also contemplated.

In one embodiment, screening for the presence of one or more alleles ofa marker on melon linkage group 10 is contemplated, such as a markerselected from the group consisting of NU0220114, NU0219125, NU0220323,NU0244737, NU0218664, NU0243568, NU0219683, and NU0219142, among others.In some embodiments, screening for an allele of a marker linked within10 cM of any of these above listed markers is also contemplated.

Screening for the presence of alleles of two or more genetic markers onlinkage group 4 or linkage group 10 may also be carried our inaccordance with the invention. Screening for the presence of one or morealleles of a marker on linkage group 4 and one or more alleles of amarker on linkage group 10 is contemplated in accordance with theinvention, Further, selecting a progeny melon plant based on thepresence of one or more alleles of such above-listed markers, or othermarker(s) linked within 10 cM of any of the above listed markers, isalso contemplated.

In certain embodiments, a method of the invention comprises identifyinga Cucumis melo plant comprising a QTL introgression on LG4 mappingbetween, and including one or more of, markers NU0219671, NU0243432,NU0219672, NU0219774, NU0220446, NU0218512, NU0243542, NU0219676,NU0220613, NU0244254, NU0220202, and NU0218915, wherein theintrogression confers elevated levels of Brix. In some embodiments amethod of the invention comprises identifying a Cucumis melo plantcomprising a QTL introgression on LG10 mapping between, and includingone or more of, markers NU0220114, NU0219125, NU0220323, NU0244737,NU0218664, NU0243568, NU0219683, and NU0219142, wherein theintrogression confers elevated levels of Brix. In other embodiments, themethod may comprise screening for an allele found in the disclosedgenetic interval of LG4 and for an allele found in the disclosed geneticinterval of LG 10. In particular embodiments, the method comprisesidentifying a Cucumis melo plant comprising a high Brix QTL on melon LG4and/or LG10. The method may also comprise crossing such a melon plantwith another plant, and may further comprise identifying a progeny plantof any generation thereof that comprises a high Brix QTL as disclosedherein.

The invention therefore allows efficient screening for, andidentification of, Cucumis melo plants and their progeny that compriseQTL conferring elevated Brix. This allows introduction of the high BrixQTL into any other genetic background capable of being bred with Cucumismelo.

The definition of these QTL allows the use of specific molecularmarkers, such as those disclosed herein, in a plant breeding program tointrogress the elevated Brix into various agronomically acceptableCucumis melo lines. Marker-assisted introgression involves the transferof a chromosomal region, defined by one or more markers, from onegermplasm to a second germplasm. An initial step in that process is thelocalization of the trait by gene mapping which is the process ofdetermining the position of a gene relative to other genes and geneticmarkers through linkage analysis. The basic principle for linkagemapping is that the closer together two genes are on the chromosome, themore likely they are to be inherited together. Briefly, a cross is madebetween two genetically compatible but divergent parents relative to atrait under study (e.g. Brix content). Genetic markers may then be usedto follow the segregation of traits under study in the progeny from thecross, often termed a “mapping population.” The current invention allowsintrogression of QTL conferring elevated Brix content. In certainembodiments of the invention, the process for producing Cucumis meloplants with increased Brix comprises introgressing at least onechromosomal locus mapping to linkage group 4 or linkage group 10 of C.melo a Cucumis melo plant having relative high Brix content into agenetic background initially having lower relative Brix content. Inspecific embodiments, the chromosomal locus comprises a locus conferringhigh Brix that is found at linkage group 4 or linkage group 10 of lineBEST.

Introgression of a particular DNA element or set of elements into aplant genotype is defined as the result of the process of backcrossconversion. A plant genotype into which a DNA sequence has beenintrogressed may he referred to as a backcross converted genotype, line,or variety. Such genotype, line, or variety may be an inbred or a hybridgenotype, line, or variety. Similarly a plant genotype lacking saiddesired DNA sequence may be referred to as an unconverted genotype,line, or variety. During breeding, the genetic markers linked to anenhanced Brix content QTL may be used to assist in breeding for thepurpose of producing Cucumis melo plants with increased Brix content. Askilled worker would understand that the introgression may be monitoredby phenotypic assays, such as of Brix content, and/or by monitoring andbreeding for the presence of molecular markers as described herein (i.e.marker assisted selection).

Localization of such markers to specific genomic regions or contigsfurther allows for use of associated sequences in breeding, to developadditional linked genetic markers, as well as to identify the mechanismfor resistance at more precise genetic and biochemical levels. It willbe understood to those of skill in the art that other markers or probeswhich more closely map the chromosomal regions as identified hereincould be employed to identify plants comprising a desired QTL for Brixcontent. Linkage blocks of various sizes could be transferred within thescope of this invention as long as the chromosomal region enhances Brixcontent. Accordingly, it is emphasized that the present invention may bepracticed using any informative molecular markers which genetically mapin similar regions, provided that the markers are polymorphic betweenthe parents.

Earliness is an important agronomic trait, as it allows growers to bringthe harvest to market sooner and reduces the risks of crop loss. Forexample, many growing areas have an off-season with weather conditionsthat are detrimental to agriculture, such as cold, rain, or reducedsunlight. By growing an early maturing hybrid, a farmer could reduce therisk that his crop will not have been harvested prior to the onset ofthese conditions. Additionally, in regions where back to back cropcycles are possible, an early harvest will allow a second planting tofit more readily within ideal growing conditions. Also, the first fruitsto market in a growing season can often fetch a premium price notavailable when the market is saturated shortly thereafter. Further,disease pressures often build up throughout a growing season and anearly harvest will avoid the more severe disease pressures that occurlate in the season or allow harvest from a field before disease symptomsfully develop and affect marketable yield from a crop.

Early development of fruit quality can also be exploited to reduce thenumber of field trips required to complete the harvest. In melons, theprimary quality attribute used to determine acceptability and grade isBrix, or total soluble solids. For example, a Brix value of 9 is highlydesirable for import to markets in the United States. For successfulmarketability, a grower cannot harvest fruit until each reachesacceptable quality levels, meaning that daily, or even twice-dailyharvest passes may be needed to get maximum yield without compromisingquality. In contrast, if the variety reaches good quality five to sevendays earlier, the grower could wait to start harvesting the first setfruit and could make fewer passes through the field to complete theharvest while maintaining fruit quality. As shown in Example 6 and FIGS.6-7, the high Brix QTL of linkage group 4 surprisingly allows forearlier accumulation of Brix to a desired level.

Flesh color is another hallmark of fruit quality which generallydevelops late in the maturation process. Further, it is known fromconsumer studies conducted in melons that consumers associate more vividcolor with greater degrees of ripeness. The presence of the Brix QTL oflinkage group 4, as found for instance in line Q4:B and as discussed inExample 6, allows for development of approximately mature orange fleshcolor earlier than expected, for instance at around 28 DPA. Further,Q4:B fruits have a deeper orange color at slip, thus the color of Q4:Bat 28 DPA is predicted to be more ripe in appearance than that of, forinstance, sister line Q4:1046 (lacking the sequences such as the BrixQTL on linkage group 4) at 37 DPA. It is especially surprising that theQ4:B line would exhibit a deeper orange color at maturity, given thatthe trait source ‘Best’ is a white-fleshed melon. However this appearsto be a desirable linkage of the association of deeper color withearly-maturing fruit.

The term “Brix” (“°Bx”) is used here to quantify the mass ratio ofdissolved solids, such as sucrose, to water in a liquid and is given inunits of degrees “°”). More specifically, a measurement of the Brixlevel of a melon fruit may be made according to methods well known inthe art, for instance by use of a saccharimeter or refractometer (e.g.Atago pocket refractometer PAL-1; Atago USA, Inc., Bellevue, Wash.). Forinstance, a measurement of 10° Bx corresponds to about 7-8 grams ofdissolved solids including sucrose per 100 grams of liquid. In certainembodiments the Brix level of such melon fruit may be, for instance, atleast 9, 9.5, or 10° Bx.

As used herein, a “female parent” refers to a Cucumis melo plant that isthe recipient of pollen from a male donor line, which pollensuccessfully pollinates an egg. A female parent can be any Cucumis meloplant that is the recipient of pollen. Such female parents can be malesterile, for example, because of genic male sterility, cytoplasmic malesterility, or because they have been subject to manual emasculation ofthe stamens. Genic or cytoplasmic male sterility can be manifested indifferent manners, such as sterile pollen, malformed or stamenlessflowers, positional sterility, and functional sterility.

As used herein, “cytoplasmic male sterility” refers to plants that arenot usually capable of breeding from self-pollination, but are capableof breeding from cross-pollination.

As used herein, “linkage” is a phenomenon wherein alleles on the samechromosome tend to segregate together more often than expected by chanceif their transmission was independent.

As used herein, a “marker” is an indicator for the presence of at leastone phenotype, genotype, or polymorphism. Markers include, but are notlimited to, single nucleotide polymorphisms (SNPs), cleavable amplifiedpolymorphic sequences (CAPS), amplified fragment length polymorphisms(AFLPs), restriction fragment length polymorphisms (RFLPs), simplesequence repeats (SSRs), insertion(s)/deletion(s) (“INDEL”(s)),inter-simple sequence repeats (ISSR), and random amplified polymorphicDNA (RAPD) sequences. DNA sequencing, e.g. of chromosomal DNA, may alsobe employed to determine the allele present at a given marker ofinterest. A marker is preferably inherited in codominant fashion (bothalleles at a locus in a diploid heterozygote are readily detectable),with no environmental variance component, i.e., heritability of 1. A“nucleic acid marker” as used herein means a nucleic acid molecule thatis capable of being a marker for detecting a polymorphism. Stringentconditions for hybridization of a nucleic acid probe or primer to amarker sequence or a sequence flanking a marker sequence refers, forinstance, to nucleic acid hybridization conditions of 1×SSC and 65° C.As used herein, “marker assay” means a method for detecting apolymorphism at a particular locus using a particular method, e.g.measurement of at least one phenotype (such as a visually detectabletrait, including disease resistance), restriction fragment lengthpolymorphism (RFLP), single base extension, electrophoresis, sequencealignment, allelic specific oligonucleotide hybridization (ASO), randomamplified polymorphic DNA (RAPD), microarray-based technologies,PCR-based technologies, and nucleic acid sequencing technologies, etc.

Desirable Cucumis melo plant traits, e.g. as displayed by agronomicallyelite lines or cultivars, and that may be independently selectedinclude, but are not limited to: plant vigor, fruit flesh color, time tomaturity, adaptation to field growth, adaptation to greenhouse growth,and resistance to one or more diseases or disease causing organisms.

Breeding of Melon Lines Displaying Elevated Levels of Brix

One aspect of the current invention concerns methods for crossing aCucumis melo line comprising a QTL conferring elevated levels of Brix asdescribed herein with itself or a second plant and the seeds and plantsproduced by such methods. These methods can be used for production andpropagation of cultivated Cucumis melo lines and hybrids displayingelevated levels of Brix without agronomically undesirable traits thathave previously been associated with the elevated Brix traits.

In accordance with the invention, novel varieties may be created bycrossing elevated Brix QTL-containing lines followed by generations ofselection as desired and inbreeding for development of uniform lines.New varieties may also be created by crossing with any second plant. Inselecting such a second plant to cross for the purpose of developingnovel lines, it may be desired to choose those plants which eitherthemselves exhibit one or more selected desirable characteristics orwhich exhibit the desired characteristic(s) when in hybrid combination.Once initial crosses have been made, inbreeding and selection aresubsequently used to produce new varieties. For development of a uniformline, often five or more generations of selfing and selection aretypical.

Uniform lines of new varieties may also be developed by way ofdoubled-haploids. This technique allows the creation of true breedinglines without the need for multiple generations of selfing andselection. In this manner true breeding lines can be produced in aslittle as one generation. Haploid embryos may be produced frommicrospores, pollen, anther cultures, or ovary cultures. The haploidembryos may then be doubled autonomously, or by chemical treatments(e.g. colchicine treatment). Alternatively, haploid embryos may be growninto haploid plants and treated to induce chromosome doubling. In eithercase, fertile homozygous plants are obtained. In accordance with theinvention, any of such techniques may be used in connection with a plantof the present invention and progeny thereof to achieve a homozygousline.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait, such as elevated levels of Brix,from one inbred or non-inbred source to an inbred that lacks that trait.This can be accomplished, for example, by first crossing a superiorinbred (recurrent parent) to a donor source (non-recurrent parent),which carries the appropriate locus or loci for the trait in question.The progeny of this cross are then mated back to the superior recurrentparent followed by selection in the resultant progeny for the desiredtrait to be transferred from the non-recurrent parent. After five ormore backcross generations with selection for the desired trait, theprogeny are heterozygous for loci controlling the characteristic beingtransferred, but are like the superior parent for most or almost allother loci. The last backcross generation would be selfed to give purebreeding progeny for the trait being transferred. In this manner therecombined alleles provided by the invention may be introgressed intoany Cucumis melo genotype.

Similarly, development of Cucumis melo varieties with improved traits byincorporation of alleles from a donor plant into an elite plant cultivarbackground can be accomplished efficiently using a method of AdvancedBackcross QTL (AB-QTL) analysis (Tanksley and Nelson, 1996), followed byfine mapping analysis. Advanced backcross QTL analysis is a breedingstrategy that allows the simultaneous identification of potentiallyuseful alleles from donor germplasm and incorporation of those allelesinto elite breeding material, using marker assisted selection. AB-QTLanalysis is accomplished through the generation of a wild x elitehybrid, followed by a series of backcrosses to the elite parent, coupledwith molecular marker and phenotypic selections. Backcross populationsare subjected to QTL analysis for desirable traits, identifying genomicregions containing useful donor alleles that are introgressed into anelite cultivar genetic background, creating near isogenic lines (NILs).Finally, the NILs and the elite parent controls are evaluated for traitsin replicated field trials (Bernacchi el al., 1998). In addition toAB-QTL analysis, subsequent fine-mapping analysis is often used topinpoint the alleles influencing the trait of interest and eliminatelinkage to undesirable alleles. This is accomplished by additionalbackcrosses, generating subNILs with reduced overlapping introgressions,that are further characterized by QTL analysis and molecular markers tomore precisely define the introgression segments contributing to desiredtraits.

The selection of a suitable recurrent parent is an Important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single locus of the recurrent variety ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Cucumis melo varieties can also be developed from more than two parents.The technique, known as modified backcrossing, uses different recurrentparents during backcrossing. Modified backcrossing may be used toreplace the original recurrent parent with a variety having certain moredesirable characteristics or multiple parents may be used to obtaindifferent desirable characteristics from each.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,herbicide resistance, resistance to bacterial, fungal, or viral disease,insect resistance, modified fatty acid or carbohydrate metabolism, andaltered nutritional quality. These comprise genes generally inheritedthrough the nucleus.

Direct selection may be applied where the single locus acts as adominant trait. Selection of Cucumis melo plants for breeding is notnecessarily dependent on the phenotype of a plant and instead can bebased on genetic investigations. For example, one can utilize a suitablegenetic marker which is closely genetically linked to a trait ofinterest. One such marker can be used to identify the presence orabsence of a trait in the offspring of a particular cross, and can beused in selection of progeny for continued breeding. This technique iscommonly referred to as marker assisted selection. Any other type ofgenetic marker or other assay which is able to identify the relativepresence or absence of a trait of interest in a plant can also be usefulfor breeding purposes.

General procedures for marker assisted selection are well known in theart. Such methods will be of particular utility in the case of recessivetraits and variable phenotypes, or where conventional assays may be moreexpensive, time consuming or otherwise disadvantageous. Types of geneticmarkers which could be used in accordance with the invention include,but are not necessarily limited to, Simple Sequence Length Polymorphisms(SSLPs) (Williams et al., 1990), Randomly Amplified Polymorphic DNAs(RAPDs), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Arbitrary Primed Polymerase Chain Reaction(AP-PCR), Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858,specifically incorporated herein by reference in its entirety), andSingle Nucleotide Polymorphisms (SNPs) (Wang et al., 1998).

Many useful traits are those which are introduced by genetictransformation techniques. The preparation of transgenes, e.g. bypreparing recombinant constructs, and genetic transformation maytherefore be used to directly insert a selected transgene into a plantof the invention or recombinant DNA sequences may, alternatively,subsequently be introduced into another genetic background bybackcrossing. Methods for the transformation of plants that are wellknown to those of skill in the art and applicable to many crop speciesinclude, but are not limited to, electroporation, microprojectilebombardment, Agrobacterium-mediated transformation and direct DNA uptakeby protoplasts.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a Cucumis melo plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a Cucumis melo plant includeone or more genes for insect tolerance, such as a Bacillus thuringiensis(B.t.) gene, pest tolerance such as genes for fungal disease control,herbicide tolerance such as genes conferring glyphosate tolerance, andgenes for quality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology or plant product(s). Forexample, structural genes would include any gene that confers insecttolerance including but not limited to a Bacillus insect control proteingene as described in WO 99/31248, herein incorporated by reference inits entirety, U.S. Pat. No. 5,689,052, herein incorporated by referencein its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, hereinincorporated by reference in their entirety. In another embodiment, thestructural gene can confer tolerance to the herbicide glyphosate asconferred by genes including, but not limited to Agrobacterium strainCP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat.No. 5,633,435, herein incorporated by reference in its entirety, orglyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No.5,463,175, herein incorporated by reference in its entirety.

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., 1991). The RNA could also be a catalytic RNA molecule (i.e., aribozyme) engineered to cleave a desired endogenous mRNA product (seefor example, Gibson and Shillito, 1997). Thus, any gene which produces aprotein or mRNA which expresses a phenotype or morphology change ofinterest is useful for the practice of the present invention.

DEFINITIONS

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided:

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions from one genetic background intoanother.

Brix: The content of total soluble solids, such as sugars and acids, ina Cucumis melo fruit, as measured by refractive index.

Cultivated Cucumis melo: Cucumis melo which is suitable for consumptionand meets the requirements for commercial cultivation. In addition tothe Cucumis melo plants themselves, and the parts thereof suitable forconsumption, such as the fruit, the invention comprises parts orderivatives of the plant suitable for propagation, Examples of partssuitable for propagation are organ tissues, such as leaves, stems,roots, shoots and the like, protoplasts, somatic embryos, anthers,petioles, cells in culture and the like. Derivatives suitable forpropagation are for instance seeds. The plants according to theinvention can be cultivated or propagated in the conventional manner butalso by means of tissue culture techniques from plant parts.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor or a chemicalagent conferring male sterility.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two non-isogenicplants.

Genotype: The genetic constitution of a cell or organism. As usedherein, “genotype” is the actual nucleic acid sequence at a locus in anindividual plant. As used herein, “phenotype” means the detectablecharacteristics (e.g. Brix content) of a cell or organism which can beinfluenced by genotype.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

LOD score: The level of confidence in an estimate of linkage distancebetween two loci.

Marker: A readily detectable phenotype or genotype, preferably inheritedin co-dominant fashion (both alleles at a locus in a diploidheterozygote are readily detectable), with no environmental variancecomponent, i.e., heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Polymorphism means the presence of one or more variations of a nucleicacid sequence at one or more loci in a population of one or moreindividuals. The variation may comprise but is not limited to one ormore base changes, the insertion of one or more nucleotides or thedeletion of one or more nucleotides. A polymorphism may arise fromrandom processes in nucleic acid replication, through mutagenesis, as aresult of mobile genomic elements, from copy number variation and duringthe process of meiosis, such as unequal crossing over, genomeduplication and chromosome breaks and fusions. The variation can becommonly found, or may exist at low frequency within a population, theformer having greater utility in general plant breeding and the lattermay be associated with rare but important phenotypic variation. Usefulpolymorphisms may include single nucleotide polymorphisms (SNPs),insertions or deletions in DNA sequence (Indels), simple sequencerepeats of DNA, sequence (SSRs) a restriction fragment lengthpolymorphism, and a tag SNP. A genetic marker, a gene, a DNA-derivedsequence, a haplotype, a RNA-derived sequence, a promoter, a 5′untranslated region of a gene, a 3′ untranslated region of a gene,microRNA, siRNA, a QTL, a satellite marker, a transgene, mRNA, dsRNA, atranscriptional profile, and a methylation pattern may comprisepolymorphisms. In addition, the presence, absence, or variation in copynumber of the preceding may comprise a polymorphism.

Poor Plant Habit: Poor plant habit is characterized by increased plantvegetative growth. A Cucumis melo plant with poor plant habit exhibitsincreased vegetative growth in comparison with a cultivated Cucumis meloplant of desirable plant habit with more defined vegetative growth.Cucumis melo plant habit is rated on a scale of 1 to 9, where 1 isacceptable plant habit and 9 is poor plant habit.

Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Recombination event: A meiotic crossing-over.

Regeneration: The development of a plant from tissue culture.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing, wherein essentially allof the morphological and physiological characteristics of a Cucumis melovariety are recovered in addition to the characteristics of the singlelocus transferred into the variety via the backcrossing technique and/orby genetic transformation.

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p=0.05) from themean.

Tissue Culture: A composition. comprising isolated cells of the same ora different type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a Cucumis melo plant by transformation.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

All references cited herein are hereby expressly incorporated herein byreference.

EXAMPLES Example 1 Development of Breeding Strategy

A breeding strategy was initiated to develop melon hybrids with improvedfruit quality such as enhanced sweetness, and in particular increasedBrix under adverse growing conditions. A Brix of 9 is often consideredto be a minimum requirement for successful marketing of melons in USmarkets, and elsewhere. This threshold Brix level can be challenging toachieve in humid environments with relatively warm nights, such asconditions commonly encountered in certain melon growing regions ofCentral and South America and elsewhere. Thus melon fruit were screenedfor high Brix within elite inbreds from distinct melon market types, asthese materials may contain beneficial alleles not found, or known to bepresent, in currently available WSH hybrids.

Potential sources of favorable alleles for Brix were identified byscreening a panel of inbred lines for high Brix in Woodland, Calif. andSalama, Guatemala, both for their performance as inbreds per se and intest crosses to WSH and Eastern type melon lines. In particular, fortyeight elite melon inbred lines chosen as having consistently high Brixwere trialed. This assembled collection consisted primarily of Asian andinodorus lines, as these distinct market types were thought to containbeneficial genetics not commonly present in WSH types, and couldtherefore be used to improve fruit quality in unrelated market types.

These materials were grown and sampled in triplicate and evaluated fortheir soluble solids in comparison to a selection of WSH inbred lines inorder to identify possible sources to achieve target Brix levels. Basedon these data, a selection of high performing lines were test crossed torecipient lines WSH 39-1046 AN and EMM 39-1, and the parents and F1progeny were assessed. Bulked fruit Brix was determined, and based ontest cross results and line performance, a line designated “BEST” wasthus identified as a high sugar inbred line that can contribute toimproved Brix level and high soluble solids. FIG. 1 illustrates theperformance of the five top lines from trials conducted at twolocations, in comparison with the Brix level of parental and controllines (WSH 39-1046 AN, WSH 39-1370 AN, and EMM 39-1), and FIG. 2illustrates data from certain test crosses to WSH 39-1046 AN.

Example 2 Recombinant Inbred Line (RIL) Development and Creation ofGenetic Map

A recombinant inbred line (RIL) population was then developed usingBEST, along with WSH 39-1046 as the low soluble solids parent.individual RILs, propagated by single seed descent, were screened inthree locations (Chile, Guatemala, and Woodland, Calif.). In addition toBrix measurements, fruit size and plant health data were collected toensure that the uncovered QTL were not pleiotropic for undesirabletraits, and to provide the highest quality phenotypic data possible;further in that disease ratings were strongly predictive of final Brixlevels. After removing outliers using a pedigree based statisticalmodel, the resulting data were used to map QTL for improved Brix.

179 RILs were then created by single seed descent from a single F1 plantfor use in quantitative genetic studies. The F3 families established bythis effort were grown in order to confirm segregation for Brix withinthe population and verify clear separation between the parents of thepopulation. These data were obtained to confirm the utility of thispopulation for mapping Brix QTL. BEST displayed an average Brix readingof 13.7 in this study, consistent with the averages of 13.7 and 15.8observed in two previous trials. The observed Brix levels were alsosignificantly higher than the low sugar parent, WSH 39-1046 AN, whichhad an average Brix of 8.6. A two tailed, heteroscedastic t-test showinga probability of significant difference of these values of 0.0068. FIG.3 provides a histogram showing the distribution of average Brix levelsin F3 families derived from line BEST. This result, combined with thebroad, approximately normal distribution of the Brix data from the F3families (FIG. 3), showed that the BEST x WSH 39-1046 AN RILs would besuitable to identify QTL from this source for subsequent breeding.

These RILs were genotyped at the F3 generation using SNP marker arrays,with a total of 1,238 polymorphic markers segregating in thispopulation, of which 1,150 were placed on the genetic map. The completedmap consisted of 13 linkage groups, ranging in size from 27.5 cM to146.1 cM, and a small cluster of ten tightly linked markers totaling 1.5cM. The average marker spacing was 1.8 cM, and the largest gap was 27.3cM. A genetic map based on such melon SNPs segregating in the cross BESTx WSH 39-1046 AN is described in Table 2. This genetic map was comparedto a separate melon genetic map, and all genetic results discussed below(linkage groups and genetic positions (cM)) refer to consensus mappositions.

TABLE 2 Linkage groups and distribution of markers used to developconsensus melon genetic map. Average Maximum distance distance LinkageLength Number of between between Consensus Group (cM) Markers markersmarkers LG 1 111.0 96 1.2 13.4 3 2 77.6 85 0.9 13.0 2 3 34.3 31 1.1 8.112 4 132.2 139 1.0 10.4 4 5 118.5 105 1.1 9.0 11 6 27.5 36 0.8 5.0 12 7119.1 114 1.1 27.3 7 8 112.6 100 1.1 11.0 9 9 84.5 103 0.8 9.0 8 10105.1 80 1.3 22.3 1 11 146.1 120 1.2 12.7 6 12 106.6 65 1.7 14.8 5 1380.7 65 1.3 18.0 10 ** 1.5 10 0.2 1.0 1 Total 1257.3 1,150 1.8 27.3 —

F4 progeny of the genotyped individuals were then tested in three triallocations for phenotypic analysis, including for Brix content. Each F4family was planted in triplicate, and eight fruit were sampled per plot.Brix data were collected and data were analyzed in order to remove biasassociated with disease gradients noted in some field locations, whichimpact overall plant vigor and thus affect Brix content as well.

Mixed model analysis revealed a significant effect of pedigree in eachlocation (P<0.001), and control of error variance was acceptable, withthe within location CVs between 9 and 15 percent. For each triallocation, outlier analysis was performed within each pedigree using thedeleted studentized residual for each line. Due to evident planthealth/disease gradients at one location, a number of statistical modelswere considered to adjust the data for non-genetic nuisance effects.Based on the mean square error of prediction (MSEP), a modelincorporating a plant health covariate, with nearest neighboradjustments to correct for micro-environmental variation was chosen.This model gave an eight percent reduction in the MSEP, and successfullyremoved the gradient in disease symptoms and Brix values (data notshown). For data from a second location a model accounting for the planthealth scores gave the best results as well. For data from a thirdlocation no further adjustments beyond the removal of outliers werepossible due to the lack of covariate data and adequate field maps.

Example 3 QTL Analysis

The least squares mean raw and adjusted Brix values for first twolocations mentioned above, and the raw values from the third location,were then used for QTL analysis using the composite interval mappingalgorithm implemented in WinCartQTL (e.g. Wang et al., 2011). Thisanalysis revealed two loci (QTL) in all three locations in both the rawand adjusted data which were derived from BEST and which contributed toelevated Brix. One identified QTL was located on linkage group four(“LG4”) initially spanning approximately 25 cM. Another locus was foundon linkage group ten (“LG10”) spanning 15 cM, and the boundaries weresubsequently refined further. FIG. 4 shows the output of the CompositeInterval Mapping (CIM) analysis for LG4 and LG10. FIG. 5 shows a portionof the melon genetic map with marker density in the region of theseloci, and indicates the 95% confidence interval for the placement ofeach locus using the adjusted Brix values from the first two locations,combined with raw values from the third growing location as discussedabove. Table 3 provides key summary statistics for each locus, alsocalculated using the adjusted values where available. The 2-LOD scorethreshold interval for QTL of LG4 is bounded by markers NU0219671(located at 42.0967 cM on consensus genetic map of FIG. 5) and NU0218915(63.54661 cM). The 2-LOD score threshold interval for QTL of LG10 isbounded by markers NU0220114 (1.670227 cM) and NU0219142 (14.3257 cM).

TABLE 3 Summary of the QTL statistics. 99% confidence intervals,additive effect sizes, and R² values as calculated in QTL Cartographer.Positions of intervals are calculated from the above described mappingpopulation. Trial 2 LOD C.I. Additive QTL Location (cM) effect R² Marker# CmBrix4_50 3 46-61 0.56 .10 37 2 42-51 0.96 .30 17 1 49-65 0.89 .27 37CmBrix10_5 3  0-15 0.41 .07 30 2  0-15 0.62 .12 30 1  0-11 0.48 .08 25

Thus the QTL analysis uncovered two loci derived from BEST whichconferred increased Brix at each trial location. These QTL may beintrogressed into parental inbred lines to provide improved fruitquality in melons, for instance as grown in Central and South Americafor export to the United States or elsewhere.

Example 4 QTL Validation: Brix and Fruit Size Phenotyping of Homozygousand Heterozygous Lines

BC2-derived lines selected to carry homo- and heterozygous alleles atthe QTL intervals of LG4 and LG10 were evaluated by collectingphenotypic data for the validation of the two melon Brix QTL, which weremapped in WSH 39-1046 AN x BEST segregating population. These QTLvalidation experiments consisted of 1 trial arranged in an RCBD designconsisting of 5 replications, and included 5 controls per replicationand a total of 100 plots, as well as 2 trials arranged in split-plotdesigns, where backcross families were the whole-plot factors and QTLgenotypes were the subplots, consisting of 5 replications, and included4 and 6 controls per replication and a total of 200 to 300 plots,respectively.

Five fruits were phenotyped per plot for Brix, flesh firmness, fruitsize (length and width), and flesh color, as well as plant health, plotvigor, fruit set (number of fruits per plot) to validate the Brix QTLand evaluate linkage drag introduced by the QTL introgression. Brix,flesh firmness and color were phenotyped using handheld refractometer,penetrometer and colorimeter, respectively, while fruit size wasevaluated as length and width of fruit cut along the stem and blossomend using digital caliper. RCBD and split plot designs were analyzedusing linear and mixed models in SAS v 9.1 (SAS Institute, Cary, N.C.,USA). All pairwise comparisons and specific within and across backcrossfamily QTL genotype comparisons were tested. In addition, the additiveand dominance effects were estimated for each backcross family. Table 4provides exemplary results of this analysis. The least square means ofQTL genotypes referred as 46/46, B/46 and H/46 correspond tointrogressions homozygous for WSH 39-1046-AN, homozygous for BEST, andheterozygous for the QTL of interest shown in the second column, whilethe genomic interval for the second QTL is fixed for WSH 39-1046-AN.Additive and dominance effects, calculated using the least square meansfor each BC-derived family, are shown on the first row of each box, andp-values of these estimates are shown on the second row. Effects weredeclared significant at the p<0.05 threshold.

TABLE 4 Comparisons of Brix phenotypes (in ° Brix) among BC-derivedlines carrying homozygous and heterozygous introgressions of BEST andWSH 39-1046-AN for the QTL on LG4 and LG10 for one representative trial.Brix Brix Brix Additive Dominance Sugar Sugar Sugar Additive DominanceIsmeans Ismeans Ismeans effect/ effect/ Ismeans Ismeans Ismeans effect/effect/ Family QTL 46/46 B/46 H/46 p-value p-value 46/46 B/46 H/46p-value p-value 1 LG 4 11.28 11.66 11.05 0.37 0.84 67.29 41.31 46.74−25.98 15.12 1 LG 4 0.37 0.26 <.0001 0.11 2 LG 4 11.12 12.25 12.27 1.13−1.18 67.69 51.04 69.76 −16.64 −20.79 2 LG 4 0.009 0.09 0.015 0.062 3 LG4 10.81 11.70 12.38 0.89 −2.24 60.49 32.92 49.47 −27.57 −5.52 3 LG 40.011 0.0006 <.0001 0.53 4 LG 4 11.15 12.87 12.45 1.72 −0.88 76.47 53.5960.70 −22.88 8.66 4 LG 4 <.0001 0.08 <.0001 0.28 5 LG 4 10.18 12.1411.63 1.96 −0.95 46.92 38.71 46.53 −8.21 −7.43 5 LG 4 <.0001 0.19 0.0780.32 1 LG 10 10.27 11.91 12.03 1.64 −1.89 61.51 87.89 81.96 26.38 −14.521 LG 10 <.0001 0.003 0.0014 0.282 2 LG 10 10.34 12.73 12.08 2.38 −1.0851.20 94.17 79.17 42.98 −12.97 2 LG 10 <.0001 0.06 <.0001 0.232 3 LG 1010.36 10.40 9.20 0.04 2.37 49.73 48.42 49.05 −1.32 0.06 3 LG 10 0.88<.0001 0.76 0.99 4 LG 10 10.65 11.48 11.50 0.83 −0.88 60.05 80.30 72.3920.25 −4.44 4 LG 10 0.032 0.19 0.002 0.68 5 LG 10 11.13 11.82 11.48 0.70−0.01 73.24 78.35 79.82 5.11 −8.05 5 LG 10 0.03 0.98 0.42 0.47

Table 5 provides exemplary results of comparisons of fruit length andwidth phenotypes for the QTL on LG4 and LC10 for one of the trials.

TABLE 5 Comparisons of fruit length and width phenotypes (in mm) amongBC-derived lines carrying homozygous and heterozygous introgressions ofBEST and WSH 39-1046-AN for the QTL on LG4 and LG10. Length LengthLength Additive Dominance Width Width Width Additive Dominance IsmeansIsmeans Ismeans effect/ effect/ Ismeans Ismeans Ismeans effect/ effect/Family QTL 46/46 B/46 H/46 p-value p-value 46/46 B/46 H/46 p-valuep-value 1 LG 4 141.58 120.30 127.25 −21.28 7.38 133.01 112.56 119.98−20.45 5.62 1 LG 4 <.0001 0.31 <.0001 0.28 2 LG 4 139.89 126.69 139.90−13.2 −13.22 135.60 117.81 131.16 −17.79 −8.92 2 LG 4 0.0009 0.03 <.00010.10 3 LG 4 134.72 112.45 125.04 −22.27 −2.92 132.73 106.92 117.59−25.82 4.46 3 LG 4 <.0001 0.73 <.0001 0.42 4 LG 4 147.55 127.42 130.98−20.13 13.01 139.86 118.12 127.13 −21.74 3.73 4 LG 4 <.0001 0.04 <.00010.44 5 LG 4 133.25 122.27 128.55 −10.98 −1.59 123.05 107.55 116.24 −15.5−1.87 5 LG 4 0.0109 0.82 <.0001 0.71

A significant increase in Brix was confirmed when the BEST allele wasintrogressed on LG4 (average 2a=1.79° Brix) in 2 of the 3 trials, aswell as significant decrease in firmness (average 2a=0.82 kgf), fruitlength (average 2a=14.18 mm), and width (average 2a=15.1 mm). Thepresence of the Brix QTL on LG10 (average 2a=1.16° Brix) was alsoconfirmed. Significant differences in Brix were observed among differentbackcross lines in all trials.

To investigate the negative association between Brix and fruit size forQTL on LG4, sugar content was calculated using assumptions on fruitshape, rind and cavity size (total fruit flesh volume equal to[((4/3)*π*((Length−10)/2)*((Width−10)/2)2)*0.82]/1000 assuming fruit isan ellipsoid, rind thickness=5 mm, seed cavity=18% fruit volume, fruitdensity=1 g/cm³) and the equation derived from historical data:[Brix=1.4*(% sugar)+0.6] (R2=0.87 as shown from our previous data). TheBEST introgression on LG4 was shown to lead to a significant decrease inthe total sugar content per fruit (average 2a=17.46 g); therefore, theincrease in Brix could be due to a decrease in fruit size. The negativeassociation between the Brix QTL on LG4 and fruit size may thus be dueto pleiotropy or close linkage with this trait. The Brix QTL on LG10 wasalso shown to increase sugar content significantly (average 2a=22.93 g).

To validate the Brix QTL on LG4 and LG10 in testcross combinations andconfirm efficacy of the two QTL in broader germplasm another trial wasconducted under commercial growing conditions during the fall of 2010.Testcrosses of BC2-derived lines carrying the introgressions of BEST orWSH 39-1046 AN at the QTL on LG4 and LG10 to 4 inbred testers(WSH-39-1066-AN, CHA-38-MONEY, WSH-39-1074-AN and ITA-39-4006-AN) wereevaluated. The trial was arranged in a split-plot design with 6replications and had a total of 96 plots. Five fruits per plot werephenotyped for Brix, firmness and fruit size (length and width). Due toearlier than expected maturation of the fruit, several plots had alreadypassed the optimal maturity stage for Brix evaluation, and data wascollected from 53% of the initially targeted number of fruits. However,results confirmed the effect of the QTL on LG4 in 3 of the 4 testcrosses(average 2aT=2.32° Brix), all apart from WSH-39-1074-AN, The effect ofthe LG4 QTL on fruit size was also confirmed. A decrease in length(average 2aT=−24.11 mm) and width (average 2aT=−23.11 mm) was found intestcrosses with all 4 testers.

To confirm the efficacy of QTL10 in different melon lines, twoadditional trials were conducted. The first trial had tester as thewhole-plot factor (WSH-39-1066-AN, WSH-39-1074-AN, ITA-39-4006-AN andCHA-38-MONEY), presence/absence of the BEST QTL10 introgressions as thesplit-plot factor, and had a total of 8 entries with replications. Fivefruit were phenotyped from each 10-plant plot for Brix, firmness andfruit size (length/width). ANOVAs, least square means, mean separationgroupings and comparisons were estimated using SAS with an appropriatemixed model for this split plot design.

Results of the first additional trial confirmed a significant increasein Brix due to the BEST introgression on QTL10 for three of the fourtesters, and therefore confirm efficacy of QTL10 in ITA, CHA andWSH-type melons (Table 6). No significant decrease in firmness wasobserved, and in the case of CHA-38-MONEY the BEST introgression onQTL10 led to significant increase in firmness. Significant increase infruit length and width was observed for the three CHA and WSH testers,but not the ITA tester (Table 6).

The second trial was designed with tester as the whole-plot factor(20070817 (ITA), WSH-39-1070-MO, CHA-192-ONTARIO-AN, GAL-188-COR-MO) andpresence/absence of the BEST QTL10 as well as QTL4 and QTL10introgressions as the split-plot factor. The trial had a total of 12entries with 15 replications. Phenotyping and data analysis wasconducted as previously described.

Results confirmed a significant increase in Brix due to the BESTintrogression on QTL10 in 20070817 and GAL-188-COR-MO, and significantincrease due to the BEST introgressions at both QTL10 and QTL4 in20070817, CHA-192-ONTARIO-AN and WSH-39-1070-AN (Table 7).

No significant decrease in firmness was observed, and in the case ofGAL-188-COR-MO the BEST introgression on QTL10 and QTL4/QTL10 led tosignificant increase in firmness. GAL-188-COR-MO and 20070817 hadsignificantly lower fruit length and width only in testcrosses withlines carrying both BEST QTL introgressions (Table 7). Significantincrease in fruit length and width due to the BEST introgression onQTL10 was only observed for the GAL tester, but not for CHA and WSH asin the previous experiment.

From the two experiments, as well as previous data on testcrosses withQTL4, it can be concluded that the efficacy of the Brix QTL has beenconfirmed for several tested melon lines (e.g. ITA, CHA, WSH and GAL forQTL10; and ITA, CHA and WSH for QTL4). QTL4 led to a decrease in fruitsize (length and width) across tested melon lines (WSH, ITA, GAL andCHA). Similarly, QTL10 led to increase in firmness (in CHA and GAL) andfruit size (in CHA, WSH, and GAL).

TABLE 6 Least square means estimates (LSM), lower and upper 95%confidence intervals of these estimates (L_ and U_95% CI), and meanseparation groupings (MSG) for all entries and traits tested. Fruit QTLBrix Firmness Length Tester LG10 LSM L_95% CI U_95% CI MSG LSM L_95% CIU_95% CI MSG LSM ITA-39- 1046 12.02 11.33 12.71 B 7.3 6.62 7.97 A 160.974006-AN ITA-39- BEST 12.95 12.27 13.64 A 7.53 6.86 8.2 A 155.54 4006-ANCHA- 1046 11.26 10.57 11.95 BC 4.87 4.2 5.55 D 156.04 38- MONE CHA- BEST13.21 12.52 13.90 A 7.79 7.12 8.47 A 164.10 38- MONEY WSH- 1046 9.568.86 10.27 D 6.11 5.41 6.80 C 158.81 39-1066 AN WSH- BEST 10.96 10.2711.65 C 6.22 5.55 6.90 BC 164.89 39-1066 AN WSH- 1046 10.74 10.05 11.43C 7.16 6.48 7.83 A 161.94 39-1074 AN WSH- BEST 10.98 10.29 11.66 C 7.076.40 7.74 AB 172.86 39-1074 AN QTL Fruit Length Fruit Width Tester LG10L_95% CI U_95% CI MSG LSM L_95% CI U_95% CI MSG ITA-39- 1046 157.06164.87 BCD 141.04 138.59 143.48 D 4006-AN ITA-39- BEST 151.77 159.32 D142.19 139.83 144.55 CD 4006-AN CHA- 1046 152.27 159.82 D 128.54 126.18130.90 F 38- MONE CHA- BEST 160.32 167.87 BC 133.90 131.54 136.26 E 38-MONEY WSH- 1046 154.91 162.72 CD 145.55 143.11 148.00 C 39-1066 AN WSH-BEST 161.11 168.67 B 154.80 152.44 157.16 AB 39-1066 AN WSH- 1046 158.16165.72 BC 151.54 149.18 153.90 B 39-1074 AN WSH- BEST 169.09 176.64 A156.29 153.93 158.65 A 39-1074 AN

TABLE 7 Least square means estimates (LSM), lower and upper 95%confidence intervals of these estimates (L_ and U_95% CI), and meanseparation groupings (MSG) for all entries and traits tested. Fruit BrixFirmness length Tester QTL LSM L_95% CI U_95% CI MSG LSM L_95% CI U_95%CI MSG LSM 20070817 1046: 1046 10.6 9.8 11.4 FG 8.2 7.6 8.7 A 195.31046: BEST 11.9 11.1 12.7 DE 7.7 7.2 8.2 AB 194.8 BEST: BEST 13.4 12.614.2 B 8.0 7.5 8.6 A 176.3 CHA- 1046: 1046 13.0 12.2 13.8 BC 7.2 6.7 7.7BC 126.9 192- 1046: BEST 13.4 12.6 14.2 B 7.3 6.7 7.8 BC 125.7 ONTARIO-BEST: BEST 14.7 13.9 15.5 A 6.8 6.3 7.4 C 127.4 AN GAL- 1046: 1046 12.111.2 12.9 CD 4.7 4.2 5.3 E 181.5 188- 1046: BEST 13.2 12.4 14.0 B 7.26.6 7.7 BC 190.7 COR- BEST: BEST 13.8 13.0 14.6 AB 6.7 6.2 7.2 C 177.6MO WSH-39- 1046: 1046 9.7 8.8 10.6 FG 5.6 5.1 6.2 D 160.7 1070-AN 1046:BEST 9.6 8.8 10.4 G 5.7 5.2 6.2 D 155.9 BEST: BEST 10.7 9.9 11.5 EF 5.44.9 5.9 DE 154.6 Fruit length Fruit width Tester QTL L_95% CI U_95% CIMSG LSM L_95% CI U_95% CI MSG 20070817 1046: 1046 191.0 199.7 A 160.2157.4 163.0 A 1046: BEST 190.5 199.1 A 160.1 157.3 162.9 A BEST: BEST172.0 180.6 B 149.7 146.9 152.5 BCD CHA- 1046: 1046 122.6 131.2 D 129.0126.2 131.8 E 192- 1046: BEST 121.4 130.0 D 130.3 127.5 133.1 E ONTARIO-BEST: BEST 123.1 131.7 D 128.4 125.6 131.2 E AN GAL- 1046: 1046 177.0186.0 B 152.9 150.0 155.8 B 188- 1046: BEST 186.3 195.0 A 161.1 158.3163.9 A COR- BEST: BEST 173.3 181.9 B 148.9 146.1 151.7 CD MO WSH-39-1046: 1046 155.9 165.6 C 151.8 148.7 154.9 BC 1070-AN 1046: BEST 151.4160.3 C 148.7 145.8 151.6 CD BEST: BEST 150.2 158.9 C 147.7 144.9 150.5D

No significant decrease in firmness was observed, and in the case ofGAL-188-COR-MO the BEST introgression on QTL10 and QTL4/QTL10 led tosignificant increase in firmness. GAL-188-COR-MO and 20070817 hadsignificantly lower fruit length and width only in testcrosses withlines carrying both BEST QTL introgressions (Table 5). Significantincrease in fruit length and width due to the BEST introgression onQTL10 was only observed for the GAL tester, but not for CHA and WSH asin the previous experiment. Thus, significant increase in Brix due tothe BEST homozygous introgression in BC2-derived lines for the QTL onLG4 (average 2a=1.79° Brix) and LG10 (average 2a=1.16 ° Brix) wasdemonstrated.

Example 5 Introgression of Brix QTL of LG4 in a Monoecious Melon Line

The Brix QTL from LG4 (i.e. “QTL04”) conferred from the donor line BESTwas associated with significant decrease in melon fruit size (average2a=−14.2 mm fruit length; average 2a=−15.1 mm fruit width) and firmness(average 2a=−0.82 kgf). According to breeding observations monoeciousmelon lines manifest a greater degree of dominance for fruit size andshape. Therefore, testcrosses of 3 monoecious lines from selected melontypes WSH-42-120-MO, HAR-173-10-4013-MO, and 20070817 (ITA) wereperformed with selected BC2F3 lines carrying or lacking the BESTintrogression on LG4 to assess the decrease in fruit size in monoeciousbackground, and explore further the possibility of deployment of theBrix QTL on LG4 in monoecious lines.

A split plot trial was designed with tester as the whole-plot factor(WSH-42-120-MO, HAR-173-10-4013-MO, 20070817) and presence/absence ofthe BEST QTL4 introgression as the split-plot factor. The trial had atotal of 6 entries with 10 replications. Five fruit were phenotyped fromeach 10-plant plot for Brix, firmness and fruit size (length/width).ANOVAs, least square means, mean separation groupings and comparisonswere estimated using SAS with an appropriate mixed model for this splitplot design.

Results showed that all testcrosses derived from a cross of a monoeciousline to a line carrying the homozygous BEST introgression on LG4 hadsignificantly higher Brix (average 2aT=2.03° Brix), lower firmness (2 of3) and lower fruit length (average 2aT=−8.44 mm) and width (average2aT=−5.35 mm) (Tables 8 and 9). A similar trial was then conducted undercommercial growing conditions, with WSH-39-1066-AN, CHA-38-MONEY,WSH-39-1074-AN and ITA-39-4006-AN as testers, and the testcross effectestimated for Brix was comparable with this experiment (average2aT=2.32° Brix), although testcross effects implied a more drasticdecrease in fruit length and width (length: average 2aT=−24.11 mm;width: average 2aT=−23.11 mm), The estimated testcross effects for fruitlength and width in this experiment demonstrate a moderate decrease infruit size, which implies that monoecious lines can “hide” the fruitsize phenotype. Thus an exemplary breeding cross would be to introgressthe QTL of LG4 into an andromonoecious male to be crossed with amonoecious female with large fruit size.

TABLE 8 Least square means estimates (“LSM”), lower and upper 95%confidence intervals of those estimates (“L_ and U_95% CI”), and meanseparation groupings (“MSG”) for tested entries and traits. Trait FruitBrix Firmness width Entry ID LSM L_95% CI U_95% CI MSG LSM L_95% CIU_95% CI MSG LSM 20070817/ 11.6 11.1 12.2 B 8.4 7.4 9.5 A 153.4 QTL4:1046 20070817/ 13.2 12.7 13.8 A 6.7 5.7 7.8 BC 148.1 QTL4: BEST HAR-173-9.5 8.9 10.0 C 8.0 7.0 8.9 AB 147.8 10-4013- MO/ QTL4: 1046 HAR-173-12.9 12.4 13.5 A 7.9 6.9 8.8 AB 141.3 10-4013- MO/ QTL4: BESTWSH-42-120- 8.6 8.1 9.2 D 6.6 5.6 7.5 B 167.9 MO/ QTL4: 1046 WSH-42-120-9.7 9.1 10.2 C 5.7 4.7 6.7 C 163.6 MO/ QTL4: BEST Trait Fruit widthFruit length Entry ID L_95% CI U_95% CI MSG LSM L_95% CI U_95% CI MSG20070817/ 150.8 155.9 C 182.4 178.6 186.3 A QTL4: 1046 20070817/ 145.7150.5 D 169.9 166.2 173.5 C QTL4: BEST HAR-173- 145.4 150.2 D 168.7165.1 172.4 C 10-4013- MO/ QTL4: 1046 HAR-173- 138.9 143.7 E 161.6 157.9165.2 D 10-4013- MO/ QTL4: BEST WSH-42-120- 165.5 170.3 A 182.2 178.5185.9 A MO/ QTL4: 1046 WSH-42-120- 161.1 166.1 B 176.6 172.8 180.5 MO/QTL4: BEST

TABLE 9 Comparisons of entries lacking and carrying the BESTintrogression at QTL4 within tester (QTL4: 1046 − QTL4: BEST is shown asDelta), lower and upper 95% confidence intervals of this estimate andp-values for all testers and traits phenotyped. Trait Brix FirmnessFruit width Fruit length P- P- P- P- Tester Delta value Lower UpperDelta value Lower Upper Delta value Lower Upper Delta value Lower Upper20070817 −1.58 0.00 −2.39 −0.76 1.66 0.00 0.68 2.64 5.26 0.00 1.76 8.7612.59 0.0.00 7.25 17.93 HAR- −3.50 0.00 −4.29 −2.70 0.1 0.82 −0.78 0.986.46 0.00 3.06 9.87 7.16 0.01 1.96 12.35 173-10- 4013-MO WSH-42- −1.020.02 −1.83 −0.21 0.92 0.05 0.00 1.85 4.34 0.02 0.84 7.84 5.58 0.04 0.2310.92 120-MO

Example 6 Introgression of Brix QTL of LG4 Confers Early BrixAccumulation

During the screening of germplasm to identify sources for increased Brixlevels, it was observed that one leading candidate, the line called‘Best’ was surprisingly also noted for the early accumulation of Brixrelative to physiological maturity (FIG. 6). As such, the strongesteffect high Brix QTL (QTL04) was tested to determine whether this locusconfers early Brix development.

A pair of sister BC2S1 lines were selected harboring the QTL withalternative allelic states at the QTL on LG4. The sister line with theQTL04 high Brix allele at the identified QTL on linkage group 4 isreferred to as “Q4:B” and that with the recurrent parent allele at theidentified QTL on linkage group 4 is referred to as “Q4:1046”. Theselines, along with the recurrent parent WSH 39-1046 AN and ‘Best’ weretransplanted in large plots, with five replicates of each line, 30plants per plot, and with three foot spacing between plants. Twoindividual female flowers were tagged at anthesis and allowed topollinate using bees. Each plant was randomly selected to have one fruitharvested at each time point post anthesis beginning at 28 days postanthesis (DPA). The starting point of 28 DPA was chosen based on theexpected physiological maturity for the recurrent parent beingapproximately 42 DPA.

Surprisingly, both BC lines matured significantly earlier than would beexpected, given the recurrent parent (FIG. 7). The line carrying thehigh Brix associated allele at the QTL on linkage group 4 (Q4:B) maturedat 33-34 DPA. This line is climacteric, and as such provides a simpleand reliable indicator of physiological maturity, since it abscises fromthe vine (“slips”), at the time point when fruit quality and texturereach their optimum. The sister line (Q4:1046) by contrast slipped at 37DPA, a full four days later than the near isogenic comparison Q4:B. Theslipping point is indicated on FIG. 7 by an arrow. However, as noted,both mature earlier than the recurrent parent, indicating additionalgenes for earliness are harbored in the background. Additionally, therecurrent parent itself had poor fruit set in this trial, and could nottherefore be evaluated past 37 DPA.

Q4:B accumulates Brix early (FIG. 7), reaching levels statisticallyindistinguishable from the final levels earlier than 28 DPA. Incontrast, Q4:1046 is still increasing in Brix at each time point in thisstudy and does not reach acceptable levels until 37 DPA. Data points inFIG. 7 represent LS Means of all samples measured for each line at eachpoint. Table 10 enumerates numbers of fruit per data point.

TABLE 10 Sample count for Brix measurements represented in FIG. 7; DPA =days post anthesis; Count = number of melons assayed. Genotype DPA CountBest 28 20 31 20 34 19 37 20 Q4: 1046 28 20 31 20 34 18 37 20 Q4: B 2820 31 19 34 19 WSH1046 28 9 31 4 34 8 37 5

Collection of data found in FIG. 7 was performed as follows: on eachharvest date, fruit were collected and for analysis on the day ofharvest. Each fruit was weighed and then cut to remove a one inch thickplanar sample half way between the blossom and stem end of the fruit.From the equatorial section, color was assessed using the Konica Minoltacolorimeter CR400. The equatorial section was then carefully peeled andhomogenized, filtered, and Brix determined using a benchtoprefractometer. This protocol differs from field based Brix testsdiscussed elsewhere in the application in that the field method relieson sampling tissue at two points along the equatorial plane with a melonbailer positioned mid way between the rind and the seed cavity. Althoughthe field and laboratory methods consistently show the samerelationships and trends, the lab based method includes more flesh fromnear the rind, and thereby results in lower Brix values by one half toone unit Brix, since melons have a rind to cavity gradient in sugaraccumulation.

Example 7 Effect on Fruit Flesh Color

In addition to determining that the QTL on linkage group 4 confersearlier physiological maturity and Brix accumulation, the color of eachfruit, and timing of coloration change, was measured, as this is anotherhallmark of fruit quality which occurs late in the maturation process.Q4:B develops approximately mature orange flesh color around 28 DPA. Theincrease in orange color intensity from 28 days to 34 days in the Q4:Bline is 2.3 using the ΔE (CIE94) color difference formula (InternationalCommission on Illumination (“CIE”); Vienna, Austria), which is designedto compare relative color differences. This difference is just above theminimum difference that can be detected by the human eye in a side byside comparison, and is not likely to be perceptibly different fromexpected mature flesh color in a non side-by-side comparison. Incontrast, a color difference of 6.1 was observed for Q4:1046, showingthat the magnitude of change in color over the last six days ofmaturation in this line is roughly three times more observable than thatof the sister line. Further, Q4:B fruits have a deeper orange color atslip, thus the color of Q4:B at 28 DPA is predicted to be more ripe inappearance than Q4:1046 at 37 DPA. Importantly, it is known fromconsumer studies conducted in melons that consumers associate more vividcolor with greater degrees of ripeness. It is especially surprising thatthe Q4:B line would exhibit a deeper orange color at maturity, giventhat the trait source ‘Best’ is a white-fleshed melon, however thisappears to be a desirable linkage of the association of deeper colorwith early-maturing fruit. This study also confirmed the relationshipbetween QTL4 and fruit size (also see Example 4 above), whereby fruitharboring Q4:B are smaller. However Q4:B fruit attain maximum size earlyin development as well, with no significant increases in size after 28DPA. In contrast, Q4:1046 continues to expand up to slip.

Example 8 Fine Mapping of Brix QTL on LG10

Trials to fine map the Brix QTL on LG10 were conducted in Yuma, Ariz.(June 2011), Woodland, Calif. (August 2011) and Honduras (March 2012).In each trial, Brix was measured in BC₂-derived lines carryingrecombination events at the targeted genomic region at the fruit harvestmaturity. Data was analyzed in IMP (SAS Software, Cary, N.C., USA) usingthe appropriate mixed model to calculate Least Square Means (LSM) of 13to 15 reps, depending on the experimental design of each trial, andLeast Significant Differences (LSD) using the student's t-test.

In Yuma, trial entries suggested that the QTL is located within theinterval of the markers NU0220114 and NU0220323 (1.67-4.48 cM), asentries Y11, Y7 and Y9 showed significantly higher Brix than entries Y12and Y8 (Table 11A). However, there were no statistically significantdifferences between the control lines (which carried the donorintrogression at the entire QTL interval) and the control lines carryingthe recurrent parent introgression. Also, three entries (not shown) werediscarded in this trial due to low germination that resulted ininsufficient data points for analysis.

Data from the trial in Woodland demonstrated a trend in agreement withthe data from the trial in Yuma. Entries W14, W10, W9 and W8 have higherBrix values than entries W6, W15, W13, W11 and W7 (Table 11B) whichimplies that the QTL is located in the interval of NU0219125 toNU0220323 (2.46-4.48 cM). Brix estimates for control entries in thistrial (entries W1-W4) were also not significantly different from eachother, but support that entries with donor introgressions at the QTLinterval had higher Brix. Two entries were removed from this trial (notshown) due to their much larger fruit size, which has an impact on theBrix of the phenotyped fruits.

The trial conducted in Honduras (Table 11C) gave further evidence thatthe QTL is within the interval of the markers NU0220114 and NU0220323(1.67-4.48 cM), as lines with the donor introgression at this region, asentries H7, H2, H5 and H3 showed significantly higher Brix than thosewith recurrent parent introgressions (entries H1, H6, and H4). Takentogether, these three trials demonstrate that the QTL interval on LG10between markers NU0220114 and NU0220323, between 1.67-4.48 cM, is ofparticular interest.

Deposit Information

A deposit of melon line BEST, which is disclosed herein above andreferenced in the claims, was made with the American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209. Thedate of deposit was Nov. 18, 2011 and the accession number for thosedeposited seeds of melon line BEST is ATCC Accession No. PTA-12263. Allrestrictions upon the deposit have been removed, and the deposit isintended to meet all of the requirements of 37 C.F.R. §1.801-1.809. Thedeposit will be maintained in the depository for a period of 30 years,or 5 years after the last request, or for the effective life of thepatent, whichever is longer, and will be replaced if necessary duringthat period.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

-   U.S. Pat. No. 5,463,175-   U.S. Pat. No. 5,500,365-   U.S. Pat. No. 5,633,435-   U.S. Pat. No. 5,689,052-   U.S. Pat. No. 5,880,275-   Bernacchi et al., Theor. Appl. Genet., 97:381-397, 1998.-   Bird et al., Biotech. Gen. Engin. Rev., 9:207, 1991.-   EP 534 858-   Gibson and Shillito, Mol. Biotech., 7:125, 1997-   Tanksley and Nelson, Theor. Appl. Genet., 92:191-203, 1996-   Wang et al., Science, 280:1077-1082, 1998.-   Wang S., et al. Windows QTL Cartographer, Dept. of Statistics, N.    Carolina State Univ., Raleigh, N.C.    (www.statgen.ncsu.edu/qtlcart/WQTLCart.htm), 2011.-   Williams et al., Nucleic Acids Res., 18:6531-6535, 1990.-   WO 99/31248

1-17. (canceled)
 18. A method of obtaining Cucumis melo germplasmcomprising the steps of: a) assaying Cucumis melo plants or a seedthereof for the presence of at least a first genetic marker geneticallylinked to a chromosomal region conferring increased Brix contentrelative to a plant lacking the region, wherein the region is selectedfrom the group consisting of a high Brix content contributing QTLdefined by flanking markers NU0219671 and NU0218915 on Cucumis melolinkage group 4, a high Brix content contributing QTL defined byflanking markers NU0220114 and NU0219142 on Cucumis melo linkage group10, and a high Brix content contributing QTL defined by flanking markersNU0220114 and NU0220323 on Cucumis melo linkage group 10; and b)selecting at least a first Cucumis melo plant or seed thereof comprisingthe genetic marker and the QTL that confers increased Brix content. 19.The method of claim 18, wherein a high Brix content contributing allelehas been inherited from melon line BEST, or a progeny of any generationthereof comprising said allele from Cucumis melo line BEST, a sample ofseed of said melon line BEST having been deposited under ATCC AccessionNumber PTA-12263.
 20. The method of claim 18, wherein said first geneticmarker is selected from the group consisting of NU0219671, NU0221090,NU0218604, NU0218684, NU0218148, NU0243432, NU0243324, NU0219095,NU0218257, NU0219354, NU0219672, NU0243607, NU0219118, NU0220372,NU0219774, NU0219889, NU0244419, NU0244478, NU0220446, NU0219650,NU0244718, NU0218943, NU0218512, NU0219136, NU0218860, NU0220597,NU0219475, NU0243542, NU0220825, NU0219448, NU0243629, NU0218829,NU0218923, NU0243807, NU0244022, NU0218871, NU0218901, NU0220173,NU0220684, NU0218887, NU0219803, NU0219004, NU0219642, NU0219025,NU0218757, NU0219015, NU0219386, NU0219676, NU0243281, NU0243478,NU0219177, NU0219316, NU0219426, NU0220839, NU0244508, NU0244665,NU0218187, NU0218513, NU0221036, NU0243535, NU0220613, NU0218306,NU0218509, NU0219086, NU0220451, NU0244507, NU0219425, NU0244254,NU0220942, NU0218848, NU0244041, NU0220202, NU0220577, and NU0218915.21. The method of claim 20, wherein said first genetic marker isselected from the group consisting of NU0219671, NU0243432, NU0219672,NU0219774, NU0220446, NU0218512, NU0243542, NU0219676, NU0220613,NU0244254, NU0220202, and NU0218915.
 22. The method of claim 21, whereinsaid first genetic marker is selected from the group consisting ofNU0219671 and NU0218915.
 23. The method of claim 18, wherein said firstgenetic marker is selected from the group consisting of NU0220114,NU0219003, NU0219125, NU0220581, NU0220762, NU0220116, NU0220849,NU0218843, NU0220323, NU0220985, NU0244737, NU0219214, NU0218908,NU0244415, NU0220766, NU0243268, NU0218664, NU0219506, NU0220650,NU0219176, NU0219728, NU0244427, NU0219075, NU0243568, NU0220301,NU0243578, NU0218206, NU0218955, NU0219589, NU0221079, NU0219683,NU0219740, NU0218191, NU0219782, and NU0219142.
 24. The method of claim23, wherein said first genetic marker is selected from the groupconsisting of NU0220114, NU0219125, NU0220323, NU0244737, NU0218664,NU0243568, NU0219683, and NU0219142.
 25. The method of claim 24, whereinsaid first genetic marker is selected from the group consisting ofNU0220114, NU0219125, NU0220323, and NU0219142.
 26. The method of claim18, wherein assaying the Cucumis melo plants or a seed thereofcomprises: assaying for a high Brix content contributing QTL defined byflanking markers NU0219671 and NU0218915 on Cucumis melo linkage group 4and a high Brix content contributing QTL defined by flanking markersNU0220114 and NU0219142 on Cucumis melo linkage group
 10. 27. The methodof claim 18, wherein assaying the Cucumis melo plants comprises PCR,single strand conformational polymorphism analysis, denaturing gradientgel electrophoresis, cleavage fragment length polymorphism analysis,TAQMAN assay, and/or DNA sequencing.
 28. A method of Cucumis melo plantbreeding comprising: a) assaying Cucumis melo plants or seeds thatproduce the plants for the presence of at least a first genetic markergenetically linked to a chromosomal region conferring increased Brixcontent relative to a plant lacking the region, wherein the region is ahigh Brix content contributing QTL defined by flanking markers NU0219671and NU0218915 on Cucumis melo linkage group 4, a high Brix contentcontributing QTL defined by flanking markers NU0220114 and NU0219142 onCucumis melo linkage group 10, or a high Brix content contributing QTLdefined by flanking markers NU0220114 and NU0220323 on Cucumis melolinkage group 10; and b) selecting at least a first Cucumis melo plantor seed that produces the plant comprising the genetic marker and theQTL that confers increased Brix; and c) crossing the first Cucumis meloplant with itself or a second Cucumis melo plant to produce progenyCucumis melo plants comprising the QTL that confers increased Brix. 29.The method of claim 28, wherein said first genetic marker is selectedfrom the group consisting of NU0219671, NU0221090, NU0218604, NU0218684,NU0218148, NU0243432, NU0243324, NU0219095, NU0218257, NU0219354,NU0219672, NU0243607, NU0219118, NU0220372, NU0219774, NU0219889,NU0244419, NU0244478, NU0220446, NU0219650, NU0244718, NU0218943,NU0218512, NU0219136, NU0218860, NU0220597, NU0219475, NU0243542,NU0220825, NU0219448, NU0243629, NU0218829, NU0218923, NU0243807,NU0244022, NU0218871, NU0218901, NU0220173, NU0220684, NU0218887,NU0219803, NU0219004, NU0219642, NU0219025, NU0218757, NU0219015,NU0219386, NU0219676, NU0243281, NU0243478, NU0219177, NU0219316,NU0219426, NU0220839, NU0244508, NU0244665, NU0218187, NU0218513,NU0221036, NU0243535, NU0220613, NU0218306, NU0218509, NU0219086,NU0220451, NU0244507, NU0219425, NU0244254, NU0220942, NU0218848,NU0244041, NU0220202, NU0220577, and NU0218915.
 30. The method of claim28, wherein said first genetic marker is selected from the groupconsisting of NU0220114, NU0219003, NU0219125, NU0220581, NU0220762,NU0220116, NU0220849, NU0218843, NU0220323, NU0220985, NU0244737,NU0219214, NU0218908, NU0244415, NU0220766, NU0243268, NU0218664,NU0219506, NU0220650, NU0219176, NU0219728, NU0244427, NU0219075,NU0243568, NU0220301, NU0243578, NU0218206, NU0218955, NU0219589,NU0221079, NU0219683, NU0219740, NU0218191, NU0219782, and NU0219142.31. The method of claim 28, further comprising the step of d) selectinga progeny plant comprising the QTL and crossing the progeny plant withitself or a third Cucumis melo plant to produce additional progenyplants.
 32. The method of claim 31, further comprising repeating step(d) about 2-10 times.
 33. The method of claim 32, wherein repeating step(d) comprises in at least one generation assaying Cucumis melo progenyplants or seed thereof for the presence of a genetic marker geneticallylinked to said QTL.
 34. The method of claim 28, wherein selecting thefirst Cucumis melo plant comprises selecting the plant based on thepresence of a genetic marker selected from the group consisting ofNU0219671, NU0243432, NU0219672, NU0219774, NU0220446, NU0218512,NU0243542, NU0219676, NU0220613, NU0244254, NU0220202, NU0218915,NU0220114, NU0219125, NU0220323, NU0244737, NU0218664, NU0243568,NU0219683, and NU0219142.
 35. The method of claim 28, wherein thechromosomal region conferring increased Brix comprises a high Brix QTLfound in Cucumis melo line BEST, wherein the QTL is defined by flankingmarkers NU0219671 and NU0218915 on Cucumis melo linkage group 4; or byflanking markers NU0220114 and NU0219142, or flanking markers NU0220114and NU0220323, on Cucumis melo linkage group 10; and wherein a sample ofseed of melon line BEST has been deposited under ATCC Accession NumberPTA-12263.
 36. The method of claim 18, wherein assaying the Cucumis meloplants comprises PCR, single strand conformational polymorphismanalysis, denaturing gradient gel electrophoresis, cleavage fragmentlength polymorphism analysis, TAQMAN assay, and/or DNA sequencing.